The Influence Of Bif-1a,Bif-1c On RABV Replication In Nerve Cells

Rabies is a fatal,progressive neurological infection.Generally,RABV first infects the peripheral nervous system,and clinical symptoms appear when the virus reaches the central nervous system.Once the disease develops,its fatality rate is almost 100%.The fact that rabies virus can infect all warm-blooded animals makes it epidemic worldwide.There are still tens of thousands of people died of rabies every year in endemic countries in Asia and Africa.Adapting strict and reasonable surveillance and diagnostic technology,and using vaccines and immunoglobulins pre-and post-exposure can effectively prevent rabies.But once clinical symptoms developes,there is little survival.It is necessary to develop effective rabies treatment drugs.Therefore,it is urgent to strengthen the study on the infectious characteristics and pathogenic mechanism of RABV in order to provide a theoretical basis for screening new therapeutic drugs and targets.Autophagy is an evolutionarily conserved degradative process in most eukaryotic cells.Autophagy is invovled in various physiological and pathological processes,including development,aging,cancer,neurodegenerative diseases and infections.Numerous studies have reported that autophagy is related to many viral pathogenic mechanisms.The ER transmembrane protein SHISA5 can interact with HCV non-structural protein 5A(NS5A),which lead to autophagic degradation and suppress viral replication;However,co-expression of ZIKV non-structural proteins NS4A and NS4B in fetal neural stem cells can decrease AKT phosphorylation and then inhibit m TOR activation,which induced autophagy and promoted viral replication.In NA cells,the RABV structural protein P can interact with BECN1 to decrease CASP2 and then activate the CASP2-AMPK-AKT-MTOR and CASP2-AMPK-MAPK signaling pathways to induce incomplete autophagy.However,what factors can cause or influence the autophagy,and what influence autophagy has on the pathogenesis of RABV is still unclear.In view of the fact that RABV infects primary neuronal cells in the natural state,primary neuronal cells was used in this study to study the relationship between Bif-1a,Bif-1c isoforms and RABV replication,and preliminarily explore autophagy and apoptosis induced by RABV in primary neuronal cells.1.The influence of RABV infection on the expression of Bif-1 in mouse primary neuronal cells.The mouse primary cerebral cortex neuron cells was isolated from 14?16 days fetal mice and cultured for 5?7days,and identified by indirect immunofluorescence.Primary neuronal cells was infected with RABV at a MOI of0.1,and virus supernatant and protein samples were harvested per 12h.Direct immunofluorescence(DFA),Western Blot,RT-q PCR and TCID₅₀ were used to detect the RABV replication in primary neuronal cells.Primary neuronal cells was infected with RABV at a MOI of 0.1,and an uninfected group was set as natural control.Total protein samples were harvested per 12h,and Western Blot was used to detect the expression of Bif-1 in primary neuronal cells infected by RABV.The results showed that the model of RABV CVS-11 strain infected mouse cerebral cortex primary neuronal cells was successfully established;and that the RABV CVS-11 strain can infect and replicate in primary neuronal cells.Western Blot results showed that compared with the control group,the expression level of Bif-1 in infection group was lower than the normal level.These results suggested that Bif-1 protein may be involved in RABV infection process.2.The influence of overexpression of Bif-1a,Bif-1c isforms in nerve cells on RABV replication.To explore the best MOI value of recombinant adenovirus infection,NA cells was infected with recombinant adenovirus Ad5-Bif-1a,Ad5-Bif-1c at MOI of0.1,1,and 5 respectively,recombinant adenovirus Adeasy-e GFP as a control group;and primary neuronal cells was infected at MOI of 10,30,and 50 respectively.NA cells and primary neuronal cells were infected at the optimal MOI value.The NA cells and primary neuronal cells was infected with recombinant adenovirus at the optimal MOI value for 12 hours and then inoculated with RABV at a MOI of 0.1.The total RNA and cell culture supernatant were harvested at 24 hpi,48 hpi and 72 hpi.The RT-q PCR and TCID50 were performed to detect the productions of viral genomic RNA and production of progeny virus respectively.The results showed the overexpression of Bif-1a?Bif-1c mediated by recombinant adenovirus had different influence on RABV replication in NA cells and primary neuronal cells;and the overexpression of Bif-1a promoted RABV CVS-11 replication and the overexpression of Bif-1c inhibited RABV CVS-11 replication in primary neuronal cells.3.The preliminary exploration of autophagy and apoptosis induced by RABV in primary neuronal cells.Primary neuronal cells was infected with RABV at a MOI of 10,and protein samples were harvested per 12 h.Western blot was used to detect the expression levels of key autophagy proteins LC3II and p62;and transmission electron microscopy was used to detect the changes of autophagosomes post RABV infection.Primary neuronal cells was infected with RABV at a MOI of 5,and protein samples were harvested every 12 hours.Western blot was used to detect the expression levels of the key apoptosis proteins Act caspase-3,Act caspase-and PARP;the results showed that RABV induced apoptosis at a MOI of 5 in primary neuronal cells.When primary neuronal cells was infected with RABV at a MOI of 10,there is no obvious transformation process from LC3 I to LC3 II,compared with the control group.There is no significant change in the number of autophagosomes in the RABV infection group.In summary,the self-established RABV infection mouse primary neuronal cell model was used in this study to research the relationship between overexpression of Bif-1a,Bif-1c and RABV replication from the perspective of the interaction between host protein and virus and preliminarily explore of autophagy and apoptosis induced by RABV in primary neuronal cells.The resluts showed that Bif-1c has a protective role on primary neuronal cells and can be used as a potential target for the development of anti-RABV drugs.